The overall goal of this proposal is to analyze the protein-nucleic acid interactions which constitute a purified chromosomal element. This basic approach is to utilize the ability to construct multi-copy extrachromosomal plasmids containing the specific gene of interest, a selectable auxotropic marker and an autonomously replicating segment of the yeast, Saccharomyces cerevisiae. Specifically, we propose to investigate two gene systems: (1) the TRP-1 gene which encodes an enzyme in the pathway of tryptophan biosynthesis and (2) the PHO-5 gene which encodes a phosphate-repressible acid phosphatase gene. Approaches are described which utilize extrachromosomal plasmids containing either gene, the genomic copy of either gene, or strains containing mutations in genes involved in the regulation of expression of either gene to aid in the investigation of the molecular mechanisms involved in nucleosomal phasing and regulation of gene expression. Specific experiments are proposed to examine several features of chromatin structure and gene expression: (1) the relationship of nucleosome phasing and DNA sequence or gene activity, (2) establishment of precisely positioned nucleosomes, (3) the protein composition and modification of a purified active or inactive chromosomal element, and (4) the presence of sequence-specific DNA binding proteins.